A variety of hormones, neurotransmitters and the like control, regulate or adjust the functions of living bodies via specific receptors located in cell membranes. Many of these receptors mediate the transmission of intracellular signals via activation of guanine nucleotide-binding proteins (hereinafter, sometimes referred to as G proteins) with which the receptor is coupled and possess the common (homologous) structure, i.e. seven transmembranes (membrane-spanning regions (domains)). Therefore, such receptors are generically referred to as G protein coupled receptors or seven transmembrane (membrane-spanning) receptors.
G protein coupled receptor proteins have a very important role as targets for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living bodies. Each molecule has its own receptor protein which is specific thereto, whereby the specificities of individual physiologically active substances, including specific target cells and organs, specific pharmacological actions, specific action strength, action time, etc., are decided. Accordingly, it has been believed that, if G protein coupled receptor genes or cDNA can be cloned, those will be helpful not only for the clarification of structure, function, physiological action, etc. of the G protein coupled receptor but also for the development of pharmaceuticals by investigating the substances which act on the receptor. Until now, only several G protein coupled receptor genes or cDNAs have been cloned but it is believed that there are many unknown G protein coupled receptor genes which have not been recognized yet.
The characteristic feature of the G protein coupled receptor proteins which have been known up to now is that seven clusters of hydrophobic amino acid residues are located in the primary structure and pass through (span) the cell membrane at each region thereof. It has been known that such a structure is common among all of the known G protein coupled receptor proteins and further that the amino acid sequences corresponding to the area where the protein passes through the membrane (membrane-spanning region or transmembrane region) and the amino acid sequences near the membrane-spanning region are often highly conserved among the receptors.
When an unknown protein has such a structure, it is strongly suggested that said protein is within a category of the G protein coupled receptor proteins. In addition, some amino acid residue alinements are common (homologous) and, by taking it as a characteristic feature, it is further strongly suggested that said protein is a G protein coupled receptor protein.
Libert, F, et al. (Science, 244:569-571; 1989) reported a method for cloning novel receptor genes by means of a polymerase chain reaction (hereinafter, sometimes referred to as PCR or a PCR technique) for a synthetic DNA primer which was synthesized based upon the information of common amino acid sequences obtained from a comparison among known G protein coupled receptor proteins. Libert, F. et al. used a, pair of synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions. However, in general, the design of primers used for the PCR regulates the molecular species of DNAs which are to be amplified. In addition, when a similarity (homology) in the amino acid sequence level is used as a basis, the use of different codons affects on the binding (hybridization) of the primer thereby resulting in a decrease in the amplifying efficiency.
Accordingly, although various novel receptor protein DNAs have been obtained using said DNA primers, it is not possible to succeed in amplifying DNAs for all receptor proteins in the prior art.
Further, the amino acid sequence which is common to from the first to the seventh membrane-spanning regions among 74 G protein coupled receptor proteins was reported by William C. Probst, et al. (DNA and Cell Biology, Vol. 11, No. 1, 1992, pp. 1-20). In this report, however, there is no suggestion for a method in which DNA coding for a novel G protein coupled receptor protein is screened by means of PCR using DNA primers which are complementary to the DNA coding for those amino acid sequences.
It would be desirable to develop DNA primers for PCR techniques which allow selective and efficient screenings of DNAs coding for the areas (regions) more nearer the full length of novel G protein coupled receptor proteins by utilizing the common (homologous) sequence(s) of the G protein coupled receptor protein or the DNA coding therefor.
It would also be desirable to develop synthetic DNA primers corresponding to the portions of the third and the sixth membrane-spanning regions, said primer being useful in screening for DNA coding for G protein coupled receptor proteins in more selective and efficient manner as compared with a series of the synthetic DNA primers corresponding to the sequences of the third to the sixth membrane-spanning regions as reported by Libert, F. et al.
G protein coupled receptor proteins are important for investigating substances which control the function of living organisms and proceeding developments thereof as pharmaceuticals. Finding and development of candidate compounds for new pharmaceuticals can be efficiently proceeded by using G protein coupled receptor proteins and by conducting receptor binding experiments and evaluating experiments on agonists/antagonists using intracellular information transmittance systems as indexes. Especially when the presence of a novel G protein coupled receptor protein can be clarified, the presence of a substance having a specific action thereon can be suggested.
If a novel DNA which codes for a novel G protein coupled receptor protein can be efficiently screened and isolated, it will now be possible to proceed with the isolation of DNA having an entire coding region, the construction of an expression system therefor and the screening of an acting ligand.
A hypothalamo-hypophysial system is one of the passages for controlling, regulating or adjusting the functions of organisms relying upon interactions of hormones and neurotransmitters with G protein coupled receptors. In the hypothalamo-hypophysial system, the secretion of pituitary hormones from the pituitary body (hypophysis) is regulated by hypothalamic hormones (hypophysiotropic releasing factors), and the functions of target cells and organs are controlled by pituitary hormones released into the blood. Functions; which are important for the living body are regulated through this system, such as maintenance of homeostasis and control of development and growth of a genital system and an individual organism. Representative examples of the hypothalamic hormones include TRH, LH-RH, CRF, GRF, somatostatin, galanin, etc. Representative examples of the pituitary hormones include TSH, ACTH, FSH, LH, prolactin, growth hormone, oxytocin, vasopressin, etc. In particular, the secretion of pituitary hormones is regulated according to a positive feedback mechanism or a negative feedback mechanism relied on the hypothalamic hormones and peripheral hormones secreted from the target endocrine glands. A variety of receptor proteins present in the pituitary gland play a major role for regulating the hypothalamo-hypophysial system.
It has been widely known that these hormones, factors and receptors are widely distributed in the brain instead of existing only locally in the hypothalamo-hypophysial system. This fact suggests that the substances which are called "hypothalamic hormones" are working as neurotransmitters or neuroregulators in the central nervous system. It is further considered that these substances are similarly distributed even in the peripheral tissues to play the role of important functions. The pancreas plays an important role of carrying out the carbohydrate metabolism by secreting not only a digestive fluid but also glucagon and insulin. Insulin is secreted from the .beta. cells and its secretion is promoted chiefly by glucose. It has, however, been known that a variety of receptors exist in the .beta. cells, and the secretion of insulin is controlled by various factors such as peptide hormones (galanin, somatostatin, gastric inhibitory polypeptide, glucagon, amylin, etc.), sugars (mannose, etc.), amino acids, and neurotransmitters in addition to glucose.
It has thus been known that in the pituitary gland and in the pancreas are present receptor proteins for many hormones and neurotransmitters, said receptor proteins; playing important roles for regulating the functions. As for the galanin and amylin, however, there has not yet been reported any discovery concerning the structure of their receptor protein cDNAs. It is not known whether there exist any unknown receptor proteins or receptor protein subtypes.
For substances regulating the functions of the pituitary gland and pancreas, there exist receptor proteins specific to said substance on the surfaces of various functional cells of the pituitary gland and pancreas. The pituitary gland and the pancreas are associations of a plurality of functional cells, and the actions of the individual substances are defined by the distributions of their target receptor proteins among the functional cells. Accordingly, a substance, in many cases, exhibits an extensive variety of actions. To comprehend such complex systems, it is necessary to clarify the relations between the acting substances and the specific receptor proteins. It is further necessary to efficiently screen for receptor protein agonists and antagonists capable of regulating the pituitary gland and pancreas, to clarify the structures of genes of receptor proteins from the standpoint of investigating and developing pharmaceuticals, and further to express them in a suitable expression system.
By utilizing the fact that a G protein coupled receptor protein exhibits homology in part of the structure thereof at the amino acid sequence level, an experiment of looking at DNAs coding for novel receptor proteins relying upon a polymerase chain reaction (hereinafter simply referred to as "PCR") has recently been made.
In the central nervous system, many receptor proteins such as dopamine receptor protein, LH-RH receptor protein, neurotensin receptor protein, opioid receptor protein, CRF receptor protein, CRF receptor protein, somatostatin receptor protein, galanin receptor protein, TRH receptor protein, etc. are G protein coupled receptor proteins, and it has been clarified that ligands to these receptors exert a variety of effects in the central nervous system.
In the immune system, an .alpha.- or a .beta.-chemokine receptor protein, an MIPI.alpha. receptor protein, an IL-8 receptor protein, a C5a receptor protein, etc. have been known as such G protein coupled receptor proteins, and are working as receptor proteins responsive to immunoregulating substances to play important roles for regulating the functions of the living body. There is, for example, an IL-6 receptor protein that acts both in the above-mentioned central nervous system and in the immune system. IL-6 is both a .beta.-cell differentiating factor and a biologically active factor related to the proliferation and differentiation of nerve cells.
It has been widely known that these hormones, factors and receptor proteins are usually widely distributed up to the peripheral tissues instead of existing only locally in the central nervous system and in the immune system and are producing important functions, respectively. Agonists and antagonists for these receptor proteins are now being developed as various useful pharmaceuticals.
For substances regulating the functions of the central nervous system and the immune system, there exist receptor proteins specific to said substance on the surfaces of various functional cells of the central nervous system and the immune system. The central nervous system and the immune system are associations of a plurality of functional cells, and the actions of the individual substances are defined by the distributions of their target receptor proteins among the functional cells. Accordingly, a substance, in many cases, exhibits an extensive variety of actions. Moreover, there is an example wherein many factors play a part in a physiological phenomenon. To comprehend such complex systems, it is necessary to clarify relations between the acting substances and the specific receptor proteins.
As discussed herein above, the G protein coupled receptor protein is present on the cell surface of living body cells and organs and has a very important role as a target for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living body cells and organs.